The present invention relates to a method for counting leukocytes contained in a specimen and to a leukocyte counter used therefor.
In a blood test, blood cell counting is frequently carried out and its clinical significance is great. For example, an erythrocyte count permits examining the presence and the degree of anemia and further permits clinical diagnosis of various cases caused by oxygen deficiency in the tissue. On the other hand, a leukocyte plays an important role in a defense function of fighting bacteria or virus entering the body, namely, an immunological function. In particular, granulocytes or leukocytes have a function of liberating protease such as elastase, etc. by the stimulation of bacteria or foreign proteins entering the body, and a function of allowing elastase etc., to respond to the bacteria or foreign proteins. Since the leukocyte count is increased or decreased in the case of many diseases, it is important to count leukocytes in a screening for diseases. In diseases requiring an early determination or cure, the leukocyte count is a particularly important examination item. Furthermore, since the leukocyte count fluctuates in accordance with ages of subjects, daytime or night-time, seasons, and other factors, the leukocyte count is therefore desired to be determined in routine medical treatments appropriately and accurately.
At present, methods for counting blood cells crudely classified into two types, i.e., a visual counting method and an automatic counting method. In the visual counting method, blood cells are counted by microscopic examination on a calculating board. The visual counting method includes a technique for counting blood cells without any treatment and a technique for counting blood cells after staining nuclei of blood cells with dye. In the automatic counting method, blood is diluted to a certain amount and then allowed to pass through a thin flow passage so as to detect and count blood cells by using electrical resistance or scattered light. In this method, a specific blood cell counter is generally used. In these counting methods, blood cells themselves are counted either by human eyes or by using a counter. However, these methods have the below mentioned problems.
In the above-mentioned visual counting method, errors in counting occur regarding the kind of cells. Such errors can be caused by erythrocytes being deficient in hemolysis or the hemagglutination of blood cells, by homogeneous broadening of leukocytes on the calculating board, or the skill of laboratory technicians. Furthermore, in the visible counting method, a nucleated cell other than a leukocyte, for example, an erythroblast cell, etc. is counted as a leukocyte. Therefore, when a nucleated cell other than a leukocyte is present in peripheral blood, it is necessary to analyze the ratio of leukocytes to the other cells by using a blood smear preparation and to correct the value counted. Moreover, the visual counting method is a manual method, and therefore it takes a long time to count cells.
In the automatic counting method, the exact counted value may not be obtained because of the appearance of erythroblast cells, deposition of fibrin, platelet agglutination, deficiency in hemolysis of erythrocytes, and the like. Moreover, some measurement devices permit classifying the counted cells by a grain size distribution measurement function or a pattern recognition. Such devices make it possible to detect diseases easily. However, such devices are expensive and large. Not only such special devices but also general devices used for counting blood cells in the automatic counting method are large and expensive. Furthermore, the maintenance is complicated, for example, cleaning of a liquid flow passage, etc. is complex. Therefore, such devices are effective for such institutes as laboratories in large-scaled hospitals or examination centers, etc., which deal with a large number of specimens. However, for medium size hospitals or practitioners, etc. that deal with a small number of specimens, the use and the maintenance of the large-size device is a heavy burden.
Therefore, objects of the present invention are a method for counting leukocytes accurately and conveniently by using a small-size and inexpensive device, and a leukocyte counter used therefor.
In order to achieve the above-mentioned object, a method for counting leukocytes of the present invention includes: liberating elastase from neutrophils, eosionphils and basophils (hereafter, these three blood cells are referred to as xe2x80x9cgranulocytesxe2x80x9d) contained in a specimen; adding an antigranulocyte elastase antibody to the thus liberated elastase; measuring the antigranulocyte elastase antibody bonded to the elastase to thereby determine the concentration of the elastase; and calculating the number of leukocytes contained in the specimen from the thus determined concentration of elastase by using a known ratio of the leukocyte count, the granulocyte count or the neutrophil count to the concentration of elastase.
Thus, the counting method of the present invention is a method for indirectly calculating the number of leukocytes by measuring the concentration of elastase contained in the granulocytes instead of actually counting leukocytes.
There are five classes of leukocytes, i.e., neutrophils, eosinophils, basophils, monocytes and lymphocytes. Three cells, i.e. neutrophils, eosinophils, basophils in all are called granulocytes. Moreover, the leukocyte count and the percentage of each class of leukocyte (a differential leukocyte count) in blood are not different between sexes and are substantially constant, although the percentage of each class of leukocyte is somewhat different between children and aged people. For example, in adult blood, the leukocyte count is about 6700 cells/xcexcl and the average percentage of each class of leukocyte is; neutrophil 55.3%, eosinophil 3.5%, basophil 0.5%, monocyte 5.0% and lymphocyte 36.6%. Furthermore, the granulocyte is the largest in number among leukocytes and contains a large amount of elastase inside. Granulocyte elastase is liberated when the granulocyte is exposed to stimulation or damage by phagocytes or inflammation, thus responding to lesion. In blood, the above-mentioned elastase is present in its deactivated state by bonding to a protease inhibitor such as xcex11-antitrypsin, or the like. The average retention time in blood is about 10 hours. Once the elastase is excreted into the tissue or urine, it does not return to the circulating blood again. Furthermore, the elastase concentration in the plasma is generally about one-several hundredth of the elastase concentration in granulocyte and is substantially negligible. The granulocyte elastase is different from elastase derived from the pancreas and an antibody for the granulocyte elastase does not cross-react with the elastase derived from the pancreas. The present invention uses such principles. More specifically, granulocytes are lysed so as to liberate the elastase from the granulocyte, and then the concentration of the liberated elastase is measured by an immunological technique. Consequently, even if the specimen is blood, the concentration can be measured exactly. From the measurement value, the number of leukocytes in the specimen can be calculated based on a known ratio of the leukocyte count, etc. to the elastase concentration. Thus, since the leukocytes are counted by using the concentration of elastase in granulocytes, the counting method is not affected by a nucleated cell other than a leukocyte, for example, an erythroblast cell, etc.; the deposition of fibrin; platelet agglutination; and deficiency in hemolysis of erythrocytes. Furthermore, the counter used for the method can be miniaturized, the cost for counting can be reduced and furthermore the counted value is not affected by the skill of laboratory technician because immunological techniques are used.
In the present invention, the leukocytes denote both individual leukocytes, i.e. neutrophils, eosinophils, basophils, monocytes and lymphocytes, and the whole population of leukocytes. Therefore, in the present invention, the number of individual blood cells constituting leukocytes, such as granulocytes including neutrophils, eosinophils and basophils, etc. may be counted, and the number of the entire population of leukocytes may be counted.
It is preferable that the method of the present invention includes bonding the same protease inhibitor as a protease inhibitor contained in the specimen to the liberated elastase, and then adding an antigranulocyte elastase antibody into the specimen. With such a preferable embodiment, when a protease inhibitor is contained in the specimen such as blood etc., the effect of the protease inhibitor can be eliminated. Therefore, it is preferable that when the specimen is whole blood, the protease inhibitor is xcex11l-antitrypsin, because xcex11-antitrypsin is present in the largest amount (about 90%) among the protease inhibitors in blood.
In the counting method of the present invention, it is preferable that a latex agglutination immunoassay be employed as the immunological technique. More specifically, it is preferable that the method of the present invention includes causing an antigen-antibody reaction between an antibody particle in which the antigranulocyte elastase antibody is bonded to a latex particle and the elastase liberated from granulocytes, and determining the degree of agglutination of the latex particle to thereby measure the antigranulocyte elastase antibody bonded to the elastase. The latex agglutination immunoassay is preferred because it is a homogeneous immunoassay in which an antigen-antibody reaction is fast, and a bound/free (B/F) separation is not required, and therefore, it includes only a few steps so as to be carried out conveniently and at low cost. In addition, the method of the present invention includes bonding the elastase liberated from granulocytes to the antigranulocyte elastase antibody bonded to a solid phase; adding a labeled antigranulocyte elastase antibody therein in this state; and measuring the labeled antigranulocyte elastase antibody bonded to the elastase. Depending upon the labels, the immunological technique may be an enzyme immunoassay, a radioimmunoassay, a fluorescence immunoassay, and the like.
In the counting method of the present invention, as the known ratios, for example, the following first and second ratios are preferably used.
The first ratio is a statistical ratio of the leukocyte count or the granulocyte count to the elastase concentration. This statistical ratio includes, for example, a regression linear expression, etc. obtained by separately measured values of the elastase concentration and the number of leukocytes, etc. in the specimen such as blood. The regression linear expression includes a regression linear expression between the elastase concentration and the granulocyte count, a regression linear expression between the elastase concentration and the number of whole leukocytes, and the like.
The second ratio is the elastase concentration with respect to one granulocyte cell. From this ratio, the number of granulocytes contained in the specimen can be calculated. The elastase concentration with respect to one granulocyte cell is generally 1 to 5 ng/ml, preferably 2 to 2.5 ng/ml. Therefore, the number of whole leukocytes in the specimen may be calculated from the number of granulocytes by using a known ratio of a granulocyte count to the number of whole leukocytes.
Moreover, as the known ratio of the leukocyte count to the elastase concentration of the present invention, the conventionally known ratio may be used, or a ratio that was calculated in advance in accordance with the kinds of specimen may be used. Furthermore, the counting of leukocytes may be carried out by either the above-mentioned visual method or the automatic counting method. Also, the method for measuring the elastase concentration is not particularly limited, however, it is preferable to use the immunological techniques employed in the present invention.
Next, the leukocyte counter of the present invention is used for performing the method for counting leukocytes according to the present invention and the method includes a means for calculating the number of leukocytes in a specimen from the concentration of elastase liberated from granulocytes in the specimen by using a known ratio of a leukocyte count, a granulocyte count or a neutrophil count to a concentration of elastase.
It is preferable that the above-mentioned leukocyte counter includes a means for liberating elastase from granulocytes in the specimen, a means for adding an antigranulocyte elastase antibody to the thus liberated elastase, a means for measuring the antigranulocyte elastase antibody bonded to the elastase to thereby determine the concentration of elastase; and a means for calculating the number of leukocytes contained in the specimen from the determined concentration of elastase by using a known ratio of a leukocyte count, a granulocyte count or a neutrophil count to the concentration of elastase.
Next, the reagent kit of the present invention is used for performing the method for counting leukocytes according to the present invention and the kit includes an antigranulocyte elastase antibody.
The reagent kit of the present invention used for the method for counting leukocytes employing the latex agglutination immunoassay of the present invention includes an antibody particle in which an antigranulocyte elastase antibody is bonded to a latex particle.
The reagent kit used for the counting method of the present invention using a labeled antibody has an antigranulocyte elastase antibody bonded to a labeled antigranulocyte elastase antibody and to a solid phase. It is preferable that the labeled antigranulocyte elastase antibody is an enzyme-labeled antigranulocyte elastase antibody.
It is preferable that the reagent kit of the present invention includes the same protease inhibitor as the protease inhibitor contained in a specimen to be determined. Furthermore, when the subject specimen is whole blood, the protease inhibitor is preferably an xcex11-antitrypsin. Furthermore, the reagent kit of the present invention includes a lysing substance.